Selected clinical studies on canine joint function and morphology using computerized gait analysis and diagnostic imaging

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THESIS

Submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD)

awarded by the University of Veterinary Medicine Hannover

by

Vladimir Galindo-Zamora (Born in Bogotá, Colombia)

Hannover 2012

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Supervisor: Prof. Dr. Ingo Nolte

Advisory Committee: Prof. Dr. Ingo Nolte

Prof. Dr. Christiane Pfarrer

Prof. Dr.-Ing. Bernd-Arno Behrens

1st Evaluation: Prof. Dr. Ingo Nolte, Klinik für Kleintiere, Stiftung Tierärztliche Hochschule Hannover.

Prof. Dr. Christiane Pfarrer, Anatomisches Institute, Stiftung Tierärztliche Hochschule Hannover.

Prof. Dr.-Ing. Bernd-Arno Behrens, Institut für Umformtechnik und Umformmaschinen Produktionstechnisches Zentrum Hannover.

2nd Evaluation: Prof. Dr. Henning Windhagen, Orthopädische Klinik der Medizinischen Hochschule Hannover.

Date of final exam: 14.08.2012

Partially funded by the National University of Colombia and the Colombian government in cooperation with the German Academic Exchange Service (DAAD) by a research scholarship awarded to Vladimir Galindo-Zamora

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Dedicated to my family

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Veterinary Surgery:

Evaluation of forelimb loads along with elbow movement and morphology in dogs before and after the arthroscopic management of unilateral medial coronoid process disease Vladimir Galindo-Zamora, Peter Dziallas, Davina C. Wolf, Sabine Kramer, Ingo Nolte, Jalal Abdelhadi, Karin Lucas, Patrick Wefstaedt

BMC Veterinary Research:

Diagnostic validity of 3 Tesla magnetic resonance imaging and digital radiographs for diagnosing stifle joint lesions in dogs with cranial cruciate ligament rupture

Vladimir Galindo-Zamora, Peter Dziallas, Davina C. Wolf, Ingo Nolte, Patrick Wefstaedt

The following manuscript is under preparation to be submitted for publication:

PLoS ONE:

Kinetic, Kinematic, Magnetic Resonance and Owner Evaluation of Dogs Before and After the Amputation of a Hind Limb

Vladimir Galindo-Zamora, Verena von Babo, Nina Eberle, Daniela Betz, Ingo Nolte, Patrick Wefstaedt

I participated as co-author in the following accepted publication:

American Journal of Veterinary Research:

Load redistribution in walking and trotting Beagles with induced forelimb lameness Jalal Abdelhadi, Patrick Wefstaedt, Vladimir Galindo-Zamora, Alexandra Anders, Ingo Nolte, Nadja Schilling

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Parts of this thesis were presented at the following meetings and conferences:

• 56. Jahreskongress der Deutsche Gesellschaft für Kleintiermedizin on October 22, 2010 in Düsseldorf, Germany (oral presentation in German).

• 57. Jahreskongress der Deutsche Gesellschaft für Kleintiermedizin on November 11, 2011 in Berlin, Germany (oral presentation in German).

• 4th Graduate School Day of the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectology, and Translational Medicine on November 26, 2011 in Bad Salzdetfurth, Germany (poster and oral presentation in English).

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Contents

1. Introduction ... 11

2 Literature review ... 19

2.1 Amputation... 19

2.2 Medial coronoid process disease... 21

2.3 Cranial cruciate ligament rupture ... 23

3. Manuscript I ... 27

3.1 Abstract ... 28

3.2 Introduction ... 28

3.3 Methods ... 30

3.3.1 Objectives ... 30

3.3.2 Patients ... 30

3.3.3 Surgical procedure... 31

3.3.4 Kinetic and kinematic gait evaluation ... 32

3.3.5 MR evaluation of the contralateral stifle joint... 34

3.3.6 Owner evaluation of patient comfort... 35

3.3.7 Ethics ... 37

3.3.8 Statistical methods... 37

3.4 Results ... 37

3.4.1 Clinical data... 37

3.4.3 MR evaluation of the contralateral stifle joint... 45

3.4.4 Owner evaluation of patient comfort... 45

3.5 Discussion ... 49

3.5.1 Limitations... 53

3.5.2 Conclusions ... 53

3.6 Competing interests... 53

3.7 Supporting information ... 53

3.8 Acknowledgements ... 53

3.9 Author contributions... 54

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Contents

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3.10 References ... 54

4. Manuscript II ... 57

4.1 Abstract ... 59

4.2 Introduction ... 60

4.3 Materials and Methods ... 61

4.3.1 Patients ... 62

4.3.2 Arthroscopy ... 62

4.3.4 Goniometric evaluation of the elbow joints ... 65

4.3.5 Radiographic and computed tomographic evaluation of the elbow joints ... 66

4.3.6 Statistical methods... 67

4.4 Results ... 67

4.4.1 Patients ... 67

4.4.3 Goniometric evaluation of the elbow joints ... 71

4.4.4 Radiographic and computed tomographic evaluation of the elbow joints ... 75

4.5.1 Limitations... 82

5. Manuscript III... 89

5.1. Abstract ... 90

5.2 Background ... 91

5.3 Materials and Methods ... 92

5.3.1 Statistical analyses... 98

5.4 Results ... 98

5.4.1 Cranial cruciate ligament damage ... 99

5.4.2 Caudal cruciate ligament damage... 99

5.4.3 Meniscal damage ... 99

5.4.4 Cartilage damage ... 99

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5.4.5 Osteoarthritis ... 100

... 101

5.6 Limitations... 106

5.7 Conclusions ... 106

5.8 Competing interests... 107

5.9 Authors’ contributions... 107

5.11 Endnotes ... 108

6. General discussion... 113

6.1 Manuscript I (Hind Limb Amputations) ... 113

6.1.1 Materials and methods... 113

6.1.2 Results ... 114

6.2. Manuscript II (Medial Coronoid Process Disease) ... 116

6.2.2 Results ... 117

6.3 Manuscript III (Cranial Cruciate Ligament Rupture)... 119

6.3.2 Results ... 120

6.4 Concluding remarks ... 123

7. Summary ... 125

8. Zusammenfassung ... 129

9. References ... 133

10. Acknowledgements ... 145

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1. Introduction

The high prevalence of orthopedic diseases in the dog makes the evaluation of functional and morphologic characteristics of joint disease in this species an important and fascinating area of research. For many years the study of lameness caused by joint disease was performed using subjective methods of evaluation, including lameness scoring systems, which have been proven to lack reliability (WAXMAN et al. 2008; BURTON et al. 2009). Besides, even the most experienced clinician has limited ability to detect subtle changes in movement (OFF and MATIS 1997a, b; GILLETTE and ANGLE 2008). Therefore, special diagnostic techniques, such as computerized (kinetic and kinematic) gait analysis, are needed to objectively and quantitatively evaluate the forces (kinetics) and movements (kinematics) involved in locomotion (DECAMP 1997; MCLAUGHLIN 2001).

Kinetic gait analysis can be used to obtain non-invasive, objective, and quantitative assessment of the forces occurring between the foot and the surface during the stance phase of the stride (MCLAUGHLIN 2001). Vertical (Fz) forces indicate the magnitude of the weight bearing and craniocaudal (Fy) forces represent breaking and propulsion forces (progression of the animal). Additionally, mediolateral (Fx) forces can be measured for each limb (BUDSBERG et al. 1987; DECAMP 1997; GILLETTE and ANGLE 2008) (Figure 1). These forces are more commonly measured in Newtons (N), and are normalized to a percentage of body weight (% BW), in order to make them comparable between animals (BUDSBERG et al. 1993; BOCKSTAHLER et al. 2007). Kinetic data allows the measurement of individual loads applied by each limb limb at the desired gate pace (walking, trotting or running).

Afterwards, the calculation of limb symmetry (symmetry index - SI) and load redistribution (LR) is possible, which have been commonly used to measure recovery and compensation in the analysis of several orthopaedic diseases, such as cranial cruciate ligament rupture (BUDSBERG et al. 1988; DECAMP et al. 1996; BÖDDEKER et al. 2012), hip dysplasia (BRADEN et al. 2004; FANCHON and GRANDJEAN 2007), and fragmented medial coronoid process (BURTON et al. 2008; BURTON et al. 2009).

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Introduction

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Figure 1. Screenshot of a gait analysis session. Fx, Fy and Fz forces detected by the force plates (in this example, only for the forelimbs).

To measure the loads, both force plates (JEVENS et al. 1993; BERTRAM et al. 2000;

MADORE et al. 2007) and instrumented treadmills (OFF and MATIS 1997b;

BOCKSTAHLER et al. 2007; BÖDDEKER et al. 2012) can be used (Figures 2 and 3).

Instrumented treadmills have many advantages, including the possibility to control important parameters such as speed, measure simultaneously all four limb forces in a reproducible way, and the need of a smaller laboratory (OFF and MATIS 1997a, b; BELLI et al. 2001;

BREBNER et al. 2006; BOCKSTAHLER et al. 2007). Besides, gait patterns are very similar when comparing the two systems (BÖDDEKER et al. 2010).

Kinematic gait analysis using high-speed infrared cameras (Figure 4) and retro-reflective markers positioned on anatomical landmarks of the canine body (Figure 3) can serve as a tool to detect and quantify changes in joint angles, step length or step velocity (HOTTINGER et al. 1996; DECAMP 1997; GILLETTE and ANGLE 2008).

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Figure 2. Example of patient walking on a runway, with a force plate embedded in it. The picture was taken in the old gait laboratory of the University of Veterinary Medicine Hannover, Foundation.

Figure 3. Example of a dog walking on an instrumented treadmill, with four independent belts. Picture taken at the gait laboratory of the University of Veterinary Medicine Hannover, Foundation. Note the retro-reflective

markers placed on the patient.

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Introduction

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The cameras detect marker movement in a 3-dimensional (3-D) space, allowing the reconstruction of the patients' movement (Figure 5). Initially, kinematics were used to characterize the normal movement pattern of different breeds (DECAMP et al. 1993;

HOTTINGER et al. 1996). More recently, the kinematic analysis of joint disease has been performed to objectively evaluate surgical success in the stifle (BÖDDEKER et al. 2012), hip (BRADEN et al. 2004), and elbow (BURTON et al. 2011), among other joints.

Figure 4: Gait laboratory of the University of Veterinary Medicine Hannover, Foundation. Note the infrared cameras (3 of 6) located around the treadmill (arrows).

Even though data obtained using kinetic and kinematic gait analysis can be correlated with other diagnotic modalities, this is carried out very seldom: only one study assessed computerized gait analysis combined with scintigraphy and computed tomography (CT) for the diagnosis of disease of the palmar sesamoid bones in dogs (MURGIA et al. 2005). In that study, CT allowed the visualization of lesions that were overlooked on plain radiographs. CT was used in one of the studies included in this thesis, as it is one of the preferred diagnostic methods to rule out different diseases, including medial coronoid process disease (MCPD) (Figure 6). CT allows multislice, multidirectional cross-sectional imaging, alleviating the problem of bone superimposition associated with radiography (MOORES et al. 2008; COOK and COOK 2009). CT also allows the evaluation of articular subchondral bone; therefore,

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lesions such as sclerosis, fissures, necrosis, cysts, and fragmentation are detectable (COOK and COOK 2009).

Figure 5. Screenshot of a gait analysis session. The 3-D perspective illustrates the retro-reflective markers, as detected by the infrared cameras.

Figure 6. Patient suffering from medial coronoid process disease (MCPD) being imaged in the CT scan (Philips Brilliance 64 CT Scanner: Philips Healthcare, Hamburg, Germany) of the Small Animal Hospital, University of Veterinary Medicine Hannover.

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Introduction

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However, the nature and extent of cartilage lesions within the joint cannot be evaluated with this diagnostic tool (MOORES et al. 2008) and other diagnostic techniques, such as magnetic resonance imaging (MRI) are therefore needed (Figure 7).

Figure 7. Patient suffering from medial coronoid process disease (MCPD) being imaged in the magnetic resonance (MR) scan (Philips Achieva 3.0T X-series MRI. Philips Healthcare, Hamburg, Germany) of the Small

Animal Hospital, University of Veterinary Medicine Hannover.

Magnetic resonance (MR) is a relatively new diagnostic imaging modality in veterinary medicine. In human orthopaedics, MRI is the preferred diagnostic tool for certain joint diseases, such as meniscal and ligament tears (CRAWFORD et al. 2007). Low-field (LF) MRI has been used and considered valuable to evaluate the appearance of normal (BAIRD et al.

1998) and pathologic stifle joints in dogs (KONAR et al. 2005b; MARTIG et al. 2006;

WINEGARDNER et al. 2007). High-field (HF) MRI has also been used to assess cartilage volume, cartilage defect and lesions on the subchondral bone of canine knees with experimentally induced osteoarthritis (OA) (BOILEAU et al. 2008). In that report, the authors concluded that MRI is useful to assess the evolution of structural changes in experimental osteoarthritis (OA); however, MR images have still limitations: one study reported that the accurate assessment of cartilage coverage of the ulnar trochlear notch (UTN) using HF MRI in post-mortem specimens was promising in mid-sized to giant breeds, but difficult in small and chondrodystrophic breeds (PROBST et al. 2008).

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The general objective of this thesis was to investigate three common clinical conditions in dogs which directly affect joint function, using a combination of diagnostic techniques in order to assess morphologic and functional changes. Thus, the specific objectives of this work were:

1. To characterize the recovery outcome of dogs undergoing a hind limb amputation. In order to evaluate the motion and weight bearing characteristics, as well as the duration of adaptation to the three-legged gait, kinematic and kinetic analyses were carried out.

Furthermore, MR images of the remaining contralateral stifle joint were made before and 4 months after amputation, in order to investigate possible changes in joint morphology. It was intended to see if there was a correlation between the functional (kinetic and kinematic gait analysis) and morphologic (MR) characteristics of the stifle joint in the contralateral hind limb, due to a hypothetic weight bearing overload of this limb. Finally, the subjective impressions of the owner with regard to the recovery of the patient were gathered and compared with the objective results from the gait analysis. The results of this part of the thesis could be useful to properly advise owners facing the decision to have their dog amputated or not.

2. To objectively evaluate forelimb load as well as elbow function and morphology before and after the arthroscopic treatment of unilateral MCPD in clinical patients, using computerized gait analysis, goniometry and radiography. CT was used to ensure that one forelimb remained healthy. Additionally, it was aimed to determine if the functional parameters (kinetic and kinematic gait analysis and goniometry) correlate with the morphologic (radiography) parameters. This part of the thesis could be useful to adequately evaluate the general belief of excellent recovery after the arthroscopic treatment of MCPD.

3. Finally, to determine the agreement between 3 T MR images and the actual surgical findings, with regard to the diagnosis of joint lesions associated with a CCLR in dogs in a clinical setting. 3 T MR is also compared to digital radiography for scoring of

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Introduction

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osteoarthritic changes. With this part of the thesis, it was aimed to confirm that the images obtained with the MR scan of the Small Animal Hospital of the University of Veterinary Medicine Hannover, Foundation, provide an accurate, non-invasive diagnosis of structural changes within the canine knee.

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2 Literature review 2.1 Amputation

The amputation of a limb is a commonly performed surgical procedure in small animal practice. It is indicated when there is a permanent and severe loss of limb function, such as severe soft tissue trauma, intractable orthopaedic conditions (mainly bone tumours and severely comminuted fractures) and financial restrictions to afford a specific treatment. Other less common conditions that might lead to an amputation include chronic osteomyelitis, neurological dysfunctions such as sciatic neuropathy or brachial plexus paralysis, congenital limb deformities, vascular disease and arteriovenous fistulas (STONE 1985; LIPOWITZ 1996; WEIGEL 2003). General contraindications of the procedure include severe orthopaedic or neurological disease affecting the remaining limbs and extreme obesity (KIRPENSTEIJN et al. 1999).

In spite of several clinical reports indicating high owner satisfaction after limb amputation in dogs (WITHROW and HIRSCH 1979; CARBERRY and HARVEY 1987; KIRPENSTEIJN et al. 1999; VON WERTHERN et al. 1999), this surgical procedure is still very critically seen by the owners, and even by some veterinarians. Particularly, owners have the tendency to think that the procedure may affect the animals emotionally, as it indeed happens in people (KIRPENSTEIJN et al. 1999; SCHULZ 2009), or that it will be disabling for the animal.

Besides, owners are often worried about the possibility of a hypothetic overload of the remaining limbs, which might lead to secondary joint pathologies. Thus, most owners are reluctant to have their dog amputated and many reject the amputation as an alternative to euthanasia or take the decision only after the patient has gone through a painful surgical and/or medical treatment process.

However, the same aforementioned clinical studies in dogs (WITHROW and HIRSCH 1979;

CARBERRY and HARVEY 1987; KIRPENSTEIJN et al. 1999; VON WERTHERN et al.

1999) have shown that most owners that were reluctant to have their dogs amputated were satisfied with the overall result. Although some behavioural problems were observed, owners considered that life quality was good.

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Literature review

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On the other hand, the fact that the owner is satisfied does not necessarily mean that the amputation does not lead to underlying structural and/or pathologic changes in the animal. In fact, one hypothesis points out that the amputation process might predispose the animal to other orthopaedic conditions; to our knowledge this hypothesis has not been confirmed, refused, or even properly evaluated.

There is one study describing weight redistribution in dogs after an amputation (KIRPENSTEIJN et al. 2000) but there are no studies investigating whether changes in kinematic parameters (such as joint angle progressions and ranges of motion) occur or not.

Furthermore, there are no studies evaluating the possible presence of morphologic changes after amputation, such as joint lesions in the remaining limbs. This lack of objective information prevents the veterinarian from providing the owners with accurate information about their concerns; besides, the veterinarian may also have his own concerns. Thus, a hesitating veterinarian might also play a role in deciding against the amputation. Even though the decision of whether to amputate a patient or not is completely up to the owner, the veterinarian is responsible for providing accurate information (WEIGEL 2003), so that the owner can take a decision he will not regret later.

Another important factor to consider is the fact that the few existing studies with amputee patients are retrospective, very likely due to the difficulty to gather enough patients to allow an accurate statistical analysis. This is due to the fact that most amputated animals are oncologic patients (VON WERTHERN et al. 1999) and owners are not always willing to allow additional examinations in a pet that may already be experiencing pain or severe systemic disease. Another important limitation in such studies is the lack of homogeneity of the populations under study. Additionally, in all these studies subjective parameters were used to evaluate the outcome of the animals. As previosly mentioned, there is only one previously published report objectively evaluating the gait of amputated dogs (KIRPENSTEIJN et al.

2000). In that study force plate analyses were carried out to measure ground reaction forces (GRF) and contact times for a population of 10 large-breed dogs which had a limb amputated (five forelimbs and five hind limbs). Additionally, the center of gravity was calculated. The

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results obtained from the amputated dogs were compared to those from 22 normal dogs of the same weight; it was found that the absence of a limb caused statistically significant changes in the GRF, impulses and contact times of the remaining limbs and the location of the animal’s centre of gravity, in comparison to the control group.

However, there are no prospective studies with animals which need to be amputated, and no study has been done to objectively evaluate kinematics (joint movement) or possible joint changes after a hind limb amputation in dogs. This information is needed in order to properly (and objectively) advise owners about the outcome of an amputation.

2.2 Medial coronoid process disease

Canine elbow dysplasia (CED) is a common disease of young large-breed dogs which may consist of one or more elbow joint disorders, namely medial coronoid process disease (MCPD), ununited anconeal process (UAP), osteochondrosis dissecans of the trochlea humeri (OCD) and joint incongruity (COOK and COOK 2009; TEMWICHITR et al. 2010). The most common condition observed in elbow dysplasia is a MCPD (TEMWICHITR et al. 2010). This disease affects more often large breeds such as Rottweilers, Labrador Retrievers and Bernese Mountain Dogs (TEMWICHITR et al. 2010) but has also been reported in medium-size and mixed-breed dogs (MEYER-LINDENBERG et al. 2002). Young dogs are more prone to the disease; however, it can also occur in older animals (VERMOTE et al. 2010).

For many years, radiography has been the standard for diagnosing CED. However, the presence and severity of MCPD and elbow incongruity can be difficult to diagnose with certainty using this imaging technique alone, due to the complex architecture of the joint and the superimposition of bone structures (REICHLE et al. 2000; COOK and COOK 2009;

FITZPATRICK et al. 2009b). In spite of this, there are many radiographic changes associated with MCPD and the secondary arthritis it generates: proximal anconeal osteophytosis, proximal radial osteophytosis and subchondral sclerosis of the semilunar notch and medial coronoid process of the ulna on flexed mediolateral and craniocaudal projections; at the anatomic location of the coronoid process, highly-indicative radiographic findings can be seen (COOK and COOK 2009).

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However, for definitive diagnosis of MCPD, other diagnostic modalities may be necessary (REICHLE et al. 2000; COOK and COOK 2009; FITZPATRICK et al. 2009b). Several studies comparing the usefulness of radiography, MR, and surgical findings (SNAPS et al.

1997), computed tomography and arthroscopy (MOORES et al. 2008), and radiography, computed tomography, and arthroscopy (AUMARM 2007) for the diagnosis of elbow dysplasia have been published. Recently, a study comparing the diagnostic value of CT and MR for the diagnosis of a FMCP was published, finding a good correlation between these diagnostic modalities and arthroscopy (KLUMPP et al. 2010). As previously mentioned in the introduction, CT is one of the preferred diagnostic methods to diagnose MCPD. CT scans provide excellent tissue differentiation without superimposition of overlying structures, as it happens with radiography (HATHCOCK and STICKLE 1993; MOORES et al. 2008; COOK and COOK 2009). Nevertheless, cartilage lesions within the joint cannot be evaluated with CT (MOORES et al. 2008); other diagnostic techniques such as MR are needed for this purpose (SNAPS et al. 1997).

Several therapeutic alternatives are possible, including medical management and arthroscopic or surgical removal of the diseased coronoid process (HUIBREGTSE et al. 1994; PUCCIO et al. 2003; TROSTEL et al. 2003; FITZPATRICK et al. 2009a). Arthroscopy is a very popular diagnostic and therapeutic method for a wide range of orthopedic conditions, including cranial cruciate ligament rupture (CCLR), osteochondritis dissecans of the humeral head (OCDH) and fragmentation of the medial coronoid process of the ulna (FMCP) (MARTINI 2003). In one study arthroscopy was found useful to detect and treat the condition even when there are no clear radiographic signs of the disease (MEYER-LINDENBERG et al. 2002). In another study, the same authors described a remarkably better postoperative outcome of the patients treated by arthroscopy, as compared with those treated by arthrotomy (MEYER- LINDENBERG et al. 2003). More recently, a literature review and meta-analysis found that the arthroscopic removal of the MCP is superior to arthrotomy and to medical treatment (EVANS et al. 2008). Besides, arthroscopy is nowadays considered the “gold standard”

technique for clinical evaluation of cartilage lesions in the canine elbow (MOORES et al.

2008) and is the method currently used at the Small Animal Hospital of the University of Veterinary Medicine Hannover, Foundation for treating patients suffering from MCPD.

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Looking at the role that computerized gait analysis might play in the study of MCPD, it is important to consider that, as opposed to other diseases, such as hip dysplasia and cranial cruciate ligament rupture, there are only few studies that look at gait characteristics in patients with MCPD. In one, joint angular, joint moment and joint power compensations of the shoulder, elbow, carpus and metacarpophalangeal (MCP) joints in dogs with unilateral lameness due to MCPD were evaluated (BURTON et al. 2008). In that study, a quantitative assessment of the effects of elbow dysplasia, specifically fragmented medial coronoid process (FMCP), on thoracic limb mechanics was performed, with the aim to define the adaptive mechanisms affecting gait in dogs with FMCP as well as facilitating an objective comparison of response to different treatment regimes for this disease. The authors suggest that multiple adaptative mechanisms occur in the affected limb in order to compensate the ongoing discomfort. However, there are no studies which prospectively evaluate forelimb kinetics or elbow joint kinematics, osteoarthritis progression or goniometry after the arthroscopic treatment of unilateral MCPD in dogs. Additionally, no controlled comparison has been made in order to determine if functional parameters, such as loads and joint angles, correlate with morphologic parameters. This knowledge is necessary to give the owners a substantiated explanation about what to realistically expect after the arthroscopic treatment of unilateral MCPD in clinical patients.

2.3 Cranial cruciate ligament rupture

One of the most common orthopedic diseases of the dog is the rupture of the cranial cruciate ligament (HAYASHI et al. 2010). This disease commonly results in hind limb lameness, as a result of joint pathologic changes such as osteoarthritis, cartilage erosion and meniscal damage (MOORE and READ 1995; INNES et al. 2000). Due to the complex and multifactorial origin of the disease, preventive strategies have not yet been developed (GRIFFON 2010).

Pathologic changes are commonly seen in the stifle joint of dogs suffering from a cranial cruciate ligament rupture (CCLR), including osteoarthritis, osteophytosis and meniscal tears (D'ANJOU et al. 2008; BÖTTCHER et al. 2010), among others. One of the most clinically relevant lesions, meniscal damage, has been reported to be present in as many as 80% of the

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cases (GAMBARDELLA et al. 1981), the medial meniscus being the most commonly affected (LAMPMAN et al. 2003).

Many different surgical techniques have been described to treat the condition. However, none of these can be considered as the gold standard (VAUGHAN 2010), and the technique performed is mainly based on the surgeon's personal preference and experience (KORVICK et al. 1994). Even though new techniques, such as tibial plateau leveling osteotomy (TPLO) and tibial tuberosity advancement (TTA) have been developed, old, "classic" techniques, including the lateral stabilization of the joint (FLO 1975), are still commonly used.

With regard to the diagnosis of joint lesions associated with CCLR, magnetic resonance (MR) imaging is the preferred diagnostic tool to evaluate internal disorders of many joints in people, including the knee (MARINO and LOUGHIN 2010). In humans, MR is commonly used to accurately diagnose certain joint diseases, such as meniscal and ligament tears (CRAWFORD et al. 2007; BLOND et al. 2008; BÖTTCHER et al. 2010). Likewise, in humans suffering from osteoarthritis, MR imaging is the modality of choice to assess the morphology of periarticular soft tissue and articular cartilage (OLIVE et al. 2010). Nevertheless, the usefulness of MR in the context of osteoarthritis, and in general of joint disease, is still not well characterized in veterinary medicine (OLIVE et al. 2010).

Low-field (LF) MR imaging has been found to be valuable in evaluating the appearance of normal and pathologic stifle joints in dogs (BAIRD et al. 1998; KONAR et al. 2005b;

WINEGARDNER et al. 2007). However, there are few studies investigating the diagnostic validity of LF MR imaging for the diagnosis of meniscal lesions in dogs with cranial cruciate ligament (CrCL) insufficiency. One of these studies found 0.3 Tesla (T) MR imaging helpful for the diagnosis of complete tears in the canine meniscus, especially in larger dogs, when compared with arthroscopy (MARTIG et al. 2006). Another study, also comparing LF MR imaging with arthroscopy, found a low accuracy of LF MR imaging (0.5 T) to identify meniscal tears (BÖTTCHER et al. 2010).

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The introduction of high-field (HF) MR magnets has significantly improved image quality and allowed accurate assessment of subchondral bone lesions, joint spaces, soft tissues, cartilage defects and osteophyte growth in canine knees with experimentally induced osteoarthritis (BOILEAU et al. 2008; D'ANJOU et al. 2008). One study compared the use of 1.5 T MR with computed radiography to assess osteophytosis, subchondral bone sclerosis, joint effusion and soft tissue thickening after experimentally induced osteoarthritis in dogs, finding MR more sensitive than radiography to detect onset and progression of osteophytosis (D'ANJOU et al. 2008). Another study investigated the sensitivity and specificity of 1.5 T MR to detect meniscal tears in clinical cases of CCLR, finding a sensitivity of 100% and a specificity of 94% (BLOND et al. 2008). However, there is no study evaluating 3 T MR images of joint surfaces, cartilage, menisci or ligaments in clinical cases of canine stifle pathology. Ideally, arthroscopy or arthrotomy should be performed, in order to directly visualize the structures of interest, and to be able to accurately assess the results of any diagnostic imaging evaluation, as has been previously described for the stifle (MARTIG et al.

2006; CRAWFORD et al. 2007; BLOND et al. 2008; BÖTTCHER et al. 2010) and the elbow (MOORES et al. 2008) in dogs, and the metacarpus in horses (OLIVE et al. 2010).

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3. Manuscript I

Kinetic, Kinematic, Magnetic Resonance and Owner Evaluation of Dogs Before and After the Amputation of a Hind Limb

Vladimir Galindo-Zamora^1,2, Verena von Babo¹, Nina Eberle¹, Daniela Betz³, Ingo Nolte¹*, Patrick Wefstaedt¹

1 Small Animal Hospital, University of Veterinary Medicine Hannover, Foundation.

Bünteweg 9, D-30559 Hannover, Germany

2 Small Animal Clinic, Faculty of Veterinary Medicine, National University of Colombia.

Carrera 30 # 45-03 (Ciudad Universitaria), Bogotá, Colombia

3 Novartis Animal Health AG, CH-4002 Basel, Switzerland

The preliminary results of this study were presented at the 56. Jahreskongress der Deutsche Gesellschaft für Kleintiermedizin on October 22, 2010 in Düsseldorf (Germany).

Partially funded by the National University of Colombia and the Colombian government in cooperation with the German Academic Exchange Service (DAAD) by a research scholarship awarded to VGZ.

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Manuscript I

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28 3.1 Abstract

This study aimed to prospectively evaluate the recovery outcome of dogs undergoing a hind limb amputation, by investigating how the animal compensates the loss of such limb, using gait (kinetic and kinematic) analyses over a four-month period. Using magnetic resonance (MR) images of the contralateral femorotibial (stifle) joint, the possible presence of morphologic changes in this joint were determined. The subjective impressions of the owner were also gathered and compared with the gait and MR analyses. Twelve patients of different breed, sex and age, in which a hind limb amputation was scheduled, were included. Kinetic data showed that 10 days after the amputation there was redistribution of the load to all remaining limbs, this load shift being more important toward the forelimbs. The recorded kinetic data showed no remarkable changes during the remaining examination time points, indicating that 10 days after the amputation patients had already established their new locomotory pattern. Kinematic data showed significant differences between sessions in the mean angle progression curves of almost all joint angles; however, the ranges of motion (ROMs) of analyzed joints were very similar before and after the amputation and remained constant in the subsequent sessions after the amputation. No changes in the signal intensity of the soft tissues evaluated in the joint were found on the MR evaluation of the contralateral stifle. Besides, no evidence of cartilage damage or osteoarthritis was seen. Finally, owners evaluated the results of the amputation very positively, both during and at the end of the study. It was concluded that dogs have a quick adaptation after a hind limb amputation, and that the adaptation process to the new locomotion begins even before the amputation is performed. This happens without evidence of morphologic changes in the contralateral stifle joint, and with a very positive evaluation from the owner.

Keywords: hind limb amputation, kinetic and kinematic analyses, magnetic resonance imaging, owner evaluation

3.2 Introduction

The amputation of a limb is a commonly performed surgical procedure in small animal practice. Severe trauma and limb tumors are the most common reasons for performing an amputation; other indications include chronic osteomyelitis, neurological dysfunctions such as

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sciatic neuropathy and brachial plexus paralysis, congenital limb deformities, vascular disease and arteriovenous fistulas [1-3].

In spite of several clinical reports indicating high owner satisfaction after limb amputation in dogs [4-7], an amputation is still very critically seen by the owners, and even by some veterinarians. Particularly, owners have the tendency to think that the procedure may affect the animals emotionally, as it indeed happens in people [7,8], or that it will be disabling for them. Besides, owners are often worried about the possibility of overload of the remaining limbs, leading to hypothetical secondary joint pathologies. Thus, many owners are reluctant to have their dog amputated, and many reject the amputation as an alternative to euthanasia or take the decision only after the patient has gone through a painful surgical and/or medical treatment process.

The lack of objective information prevents the veterinarian from providing the owners with accurate information about these concerns. A hesitant veterinarian might then play a role in the owner deciding against the amputation. There is only one previously published report objectively evaluating the gait of amputated dogs [9]. In that study, force plate analyses were carried out to measure ground reaction forces (GRF) and contact times in a population of 10 large-breed dogs which had a limb amputation (five forelimbs and five hind limbs).

Additionally, the center of gravity was calculated. It was found that the absence of a limb caused statistically significant changes in the GRF, impulses and contact times of the remaining limbs and the location of the animal’s centre of gravity, in comparison to a control group of 22 healthy dogs. However, there are no prospective studies with animals which need to be amputated, and no study has been performed objectively evaluating kinematics (joint movement) or possible joint changes after a hind limb amputation in dogs. This information is needed in order to properly (and objectively) advise owners about the outcome of an amputation.

The general aim of the present study was therefore to prospectively evaluate the gait in dogs before and after amputation of a hind limb, both objectively and subjectively, in order to describe how the animal compensates the loss of a hind limb. Additionally, the stifle of the remaining limb was evaluated for the presence of possible morphologic changes, using magnetic resonance (MR) imaging, due to a hypothetic weight bearing overload of this limb.

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The results of this study could be useful for properly advising owners facing the decision to have their dog amputated or not.

3.3 Methods 3.3.1 Objectives

The main objective of the present study was to characterize the recovery outcome of dogs undergoing a hind limb amputation. In order to evaluate the motion and weight bearing characteristics, as well as the duration of adaptation to the three-legged gait, kinematic and kinetic analyses were carried out. Furthermore, magnetic resonance (MR) images of the remaining contralateral femorotibial (stifle) joint were made before and 4 months after the amputation, in order to investigate possible changes in joint morphology. We intended to see if there was a correlation between the functional (kinetic and kinematic gait analysis) and morphologic (MR) characteristics of the stifle joint in the contralateral hind limb. Finally, the subjective impressions of the owner with regard to the recovery of the patient were gathered and compared with the objective results from the gait analysis.

It was hypothesized that there would be marked changes both in the kinetic and the kinematic parameters after the amputation, but that those changes would not impair the ability of the animal to lead a normal life. Based on our clinical experience and some of the aforementioned studies [4-7], it was also hypothesized that there would not be any changes in the contralateral stifle on the MR examination. Thus, after the initial reluctance to the amputation, owners would be satisfied with the procedure.

3.3.2 Patients

All dogs presented at the Small Animal Hospital of the University of Veterinary Medicine Hannover, Foundation (Germany), between March 2010 and October 2011, which needed a hind limb amputation, were included in the study. In total, 12 patients were enrolled. Two additional patients were not included due to aggressiveness in one case, and presence of metallic orthopedic implants in both knees, making it unadvisable to perform the MR, in the other case.

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Before surgery a thorough physical examination, including all remaining limbs and the spine, was performed to rule out any disease which might obscure the results. This examination was repeated 10, 30, 90 and 120 days after the amputation. It was planned that, in case an abnormality was suspected, all necessary diagnostic examination tools would be used to determine the type and location of such an abnormality and its possible relationship with the amputation.

3.3.3 Surgical procedure

On the arthroscopy day, all animals were considered good anesthetic candidates (physical status 2 according to the American Society of Anesthesiologists classification system), based on the general clinical examination and blood work. The animals were premedicated using a combination of levomethadone (0.6 mg/kg, L-Polamivet®: Intervet Deutschland GmbH, Unterschleißheim, Germany) and diazepam (0.5 mg/kg, Diazepam-ratiopharm®: Ratiopharm GmbH, Ulm, Germany); anesthesia was induced with propofol dosed to effect (1-4 mg/kg, Narcofol® 10 mg/mL: CP-Pharma Handelsgesellschaft GmbH, Burgdorf, Germany). After orotracheal intubation, anesthesia was maintained with isoflurane (Isofluran CP®: CP-Pharma Handelsgesellschaft GmbH, Burgdorf, Germany) in a 1:1 oxygen:air mixture adjusted according to the clinical signs of anesthetic depth (end-tidal isoflurane 0.7-1.5 vol%) and a continuous rate infusion (CRI) of fentanyl (0.16 µg/kg/min, Fentanyl-Janssen® 0.05 mg/mL:

Janssen-Cilag GmbH, Neuss, Germany), lidocaine (50 µg/kg/min, Xylocain® 2%:

AstraZeneca GmbH, Wedel, Germany) and ketamine (10 µg/kg/min, Ketamin 10%:

Selectavet Dr. Otto Fischer GmbH, Weyarn-Holzolling, Germany). Additionally, a preoperative epidural anesthesia with bupivacaine (0.5 mg/kg, Bupivacain-RPR-actavis®

0.5%: Actavis Deutschland GmbH & Co. KG, Langenfeld, Germany) and morphine (0.1 mg/kg, Morphin Hexal® 10 mg/mL: Hexal AG, Holzkirchen, Germany) and a intraoperative sciatic nerve block with lidocaine (1 mg/kg, Xylocain® 2%, AstraZeneca GmbH, Wedel, Germany) were performed. For postoperative analgesia, carprofen (4 mg/kg, Rimadyl®

Injektionslösung: Pfizer GmbH, Berlin, Germany) and the aforementioned CRI of fentanyl, lidocaine and ketamine were used.

The surgical procedure was performed by disarticulation of the hip, as described elsewhere [2]. Depending on their clinical status, the dogs remained in the hospital for approximately 5 days.

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Kinetic (forces) and kinematic (movement) gait analysis was performed one to three days before the amputation, as well as 10, 30, 90 and 120 days after surgery. Kinetics were measured using a specially designed treadmill (Treadmill model 4060-80: Bertec Corporation, Columbus, OH, USA) consisting of four separate belts, each of them with an integrated force plate underneath. This design allowed the simultaneous measurement of all limb forces.

Kinematic analysis was performed with the aid of retro-reflective markers (Ø 16 mm reflective markers: Vicon Motion Systems Ltd., Oxford, UK) positioned on 24 anatomic landmarks (8 per remaining limb), using double-sided adhesive tape; the location of these markers has been previously described [10,11] and is illustrated in Figure 1. Six high-speed infrared cameras (MX3+ camera system: Vicon Motion Systems Ltd., Oxford, UK) were used to record marker movement in all three remaining limbs simultaneously, as the animals were walking at a controlled speed (measurement frequency: 100 Hz). Before each measurement, static and dynamic camera calibration was performed using an L-shaped calibration device (Vicon Calibration Device: Vicon Motion Systems Ltd., Oxford, UK).

On each gait analysis session, patients were gently introduced to the gait on the treadmill; on the first day, a speed at which each individual patient walked comfortably on the treadmill was determined; on each subsequent session the patient was evaluated using the same speed, ranging from 0.5 to 0.8 m/s. During each gait analysis session, two to six trials were recorded, each with a duration of approximately 30 seconds, until at least one valid trial was obtained. A valid trial was defined as 10 consecutive regular steps, in which the dog walked smoothly, without any external forces from the handler being applied, with all paws landing on the appropriate force plate, without overstepping. Video recording was performed, to ensure that the steps were appropriate for analysis.

Both kinetic and kinematic data were simultaneously recorded using commercially available software (Vicon Nexus: Vicon Motion Systems Ltd., Oxford, UK).

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Figure 1. Localization of retro-reflective markers and measured angles.

A. Example of the localization of the retro-reflective markers on a healthy patient; B. Illustration of the localization of the retro-reflective markers on the anatomical reference points and the measured angles.

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Ten consecutive steps were afterwards analyzed for the following kinetic parameters: peak vertical force (PFz), mean vertical force (MFz), and vertical impulse (IFz). All forces were normalized to the individual body weight of each dog and data were expressed as percentage of body weight (% BW). Mean ± standard deviation (SD) was calculated from 10 valid consecutive steps. Afterwards, load redistribution (LR) was calculated for each measured parameter (PFz, MFz, IFz) using the following equation (according to Steiss et al. [12]): % load bearing = Fz of the limb/total Fz of all limbs*100. The kinetic data were processed using commercial software (MyoResearch XP Master Edition, Noraxon U.S.A. Inc., Scottsdale, AZ, USA) and exported to a Microsoft® Excel 2007 spreadsheet.

In order to process the kinematic data in Vicon Nexus, all markers were labeled in a trial.

Then, 10 valid foot strikes were marked manually to define the gait cycle (stance and swing phases) of each limb. Using a 2-dimmensional (2-D) model, projected flexion and extension angles of each remaining joint were calculated: contralateral (with respect to the amputated hind limb) scapulohumeral joint, contralateral cubital joint, contralateral carpal joint, ipsilateral (with respect to the amputated hind limb) scapulohumeral joint, ipsilateral cubital joint, ipsilateral carpal joint, contralateral coxofemoral joint, contralateral femorotibial joint and contralateral tarsal joint. Measured angles are illustrated in Figure 1. In order to compare the movement pattern of each analyzed joint, the gait cycles were normalized to 100 in all dogs and displayed as percentage of one whole stride. The mean joint angle and the range of motion (ROM) of the aforementioned joints were calculated from the mean joint angle progression curves calculated from the 10 strides per dog. The kinematic data were processed using commercial software (Vicon Nexus and Bodybuilder: Vicon Motion Systems Ltd., Oxford, UK) and then exported to a Microsoft® Excel 2007 spreadsheet.

3.3.5 MR evaluation of the contralateral stifle joint

The MR examination was performed under general anesthesia before and 120 days after amputation. The anesthetic protocol was the same described above, excluding local anesthetics and CRIs. Using a state-of-the-art 3 T MR scan(Philips Achieva 3.0T X-series MRI: Philips Healthcare, Hamburg, Germany), images were obtained from the contralateral

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knee, with the dog positioned in lateral recumbency; the limb to be examined was in a non- dependent position, with the joint at an angle of ~ 135^o. Small (11 cm Ø) surface ring coils (Achieva 3.0T Musculoskeletal SENSE Flex S coil 2 elements) as image enhancers were used, positioned parallel to each other, lateral and medial to the affected knee, and with the joint centered between the two coils. The MR protocol used included a 3-D (3-dimensional) PDW (proton-density weighted) acquisition sequence, which was afterwards reconstructed in sagittal, dorsal and transversal planes, a PDW HR (high-resolution) TSE (turbo spin echo) SENSE (sensitivity encoding for fast MR) sequence in sagittal plane, a PDW HR SPAIR (spectrally adiabatic inversion recovery) SENSE in sagittal plane and a T1-weighted TSE clear (constant level appearance) sequence in sagittal plane (Table 1). This protocol had been previously standardized and considered suitable for performing in clinical cases, as image quality is good and acquisition time is only 20 minutes (total examination time is about 40 minutes including positioning, reference scan, survey, and sequence planning).

Using a high-resolution diagnostic screen (EIZO RadiForce™ RX211 Medical color LCD monitor: Enzo Nanao Corporation, Hakusan, Ishikawa, Japan) the images were assessed by a trained evaluator (VGZ), who looked for changes in the signal intensity of the cranial cruciate ligament (CrCL), the caudal cruciate ligament (CdCL) and the lateral and medial menisci.

Possible changes in the cartilage surfaces, as well as evidence of osteoarthritic changes were also evaluated in the lateral and medial femoral condyles, femoral trochlear groove, patella and tibial plateau.

It was expected that, due to a possible underlying metastatic disease, some patients could die or be euthanized before the end of the study; if that was the case, it was planned to ask the owner to authorize the MR examination postmortem.

3.3.6 Owner evaluation of patient comfort

The owner was requested to fill out an evaluation form (modified from Hielm-Björkman et al.

[13]) before the amputation and 10, 30, 90 and 120 days after the procedure, in order to gather his/her (subjective) impressions with regard to patient comfort and recovery; these results were compared to those (objective) of the gait analysis.

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Table 1. Magnetic resonance imaging sequences used in this study and their parameters

Sequence Plane TR TE Slice

(mm)

Gap (mm)

FOV (mm) Flip angle

Matrix Orientation

PDW 3-D 1300 34 100x100x70 220x167 Joint centered

PDW Sagittal 2 90° True sagittal

PDW Dorsal 2 90° Parallel to patella ligament

PDW Transverse 2 90° Parallel to tibial plateau

PDW HR aTSE SENSE Sagittal 4326 30 2 0.2 120x120x48 90° 480x296 True sagittal PDW HR SPAIR SENSE Sagittal 4701 30 2 0.2 800x800x46 90° 228x160 True sagittal T1-weighted TSE clear Sagittal 665 18 1.8 0.18 90x90x39 90° 180x134 True sagittal

TR: Repetition time; TE: Echo Time; FOV: Field of view; PDW: proton-density weighted; 3-D: 3-dimensional; HR: high resolution; TSE: turbo spin echo;

SENSE: sensitivity encoding; SPAIR: spectrally adiabatic inversion recovery; clear: constant level appearance

Table 2. Patients included in this study

Patient Breed Sex Age Weight Reason to amputate Gait analyses PO MR

(Years) (kg) Pre 10 30 90 120

1 Boxer Male 8 32 Osteosarcoma + + + + + +

2 Labrador Female 3 31 Rhabdomyosarcoma + + + + + +

3 Mixed-breed dog Female 4 32 Osteosarcoma + + + + + +

4 Mixed-breed dog Male 1 20 Severe soft tissue trauma + + + + + +

5 Mixed-breed dog Male 12 31 Osteosarcoma + + + E - -

6 Swiss Mountain dog Female 10 39 Osteosarcoma + + + + E -

7 Bernese Mountain dog Male 2 40 Femoral fracture nonunion - - - +

8 German Shepherd mix Male 7 26 Severe soft tissue trauma + + E - - -

10 Mixed-breed dog Female 11 8 Malignant sarcoma + + + + + +

11 Landseer Female 2 54 Fibrosarcoma + + + + + -

PO MR: Postoperative MR scan; +: Performed; -: Not performed; E: Euthanasia

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At the end of the study (day 120), owners filled out a questionnaire to assess their final impression regarding the degree of activity and life quality of the dog, and their general impression of and satisfaction with the procedure; besides, owners were encouraged to make further comments. It was planned that if the animal died before the end of the study, an appropriate moment would be looked for to ask the owner to fill out the questionnaire. The questions of the questionnaire were adapted from Carberry and Harvey [4], Withrow and Hirsch [5], von Werthern et al. [6] and Kirpensteijn et al. [7].

3.3.7 Ethics

This study was carried out in accordance with the German Animal Welfare Guidelines and was approved by the Ethics Committee of the Lower Saxony State Office for Consumer Protection and Food Safety (Approval Number: 10A071). Besides, all owners agreed to their pets taking part in the study and signed a consent form.

3.3.8 Statistical methods

Due to the small sample size and very heterogeneous patient population included in this study, it was decided to use non-parametric statistics. Thus, data were analyzed using a Kruskal- Wallis one-way ANOVA test to compare medians between sessions; when statistically significant differences were found, a Wilcoxon signed-rank test for paired observations was performed to determine which session was different. All tests were considered statistically significant if p<0.05 and were performed using standard statistical software (GraphPad Prism® Version 4: GraphPad Software, Inc. La Jolla, California, USA). Descriptive statistics were calculated using Microsoft® Excel 2007, where appropriate.

3.4 Results

3.4.1 Clinical data

Breed, sex, age, reason to amputate and performed evaluations of the 12 patients enrolled in this study are illustrated in Table 2. As can be seen in this table, the most common reason for performing the amputation was a tumor, followed by trauma and one surgical complication.

Six right and six left hind limbs were amputated. Patient 8 died unexpectedly soon after the amputation due to abdominal bleeding caused by a previously asymptomatic hepatic hemangiosarcoma. Nine patients survived until the end of the study.

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Patient 12 presented bilateral hip osteoarthritis; however, it was asymptomatic, and no clinical signs (pain, lameness, difficulty standing up, etc.) were detected before or after the amputation. All other patients showed no abnormalities in the clinical examination of the remaining limbs. No patient showed spine abnormalities throughout the study.

3.4.2 Kinetic and kinematic gait evaluation

The results of the kinetic and kinematic evaluations are presented in Figures 2 to 5 and Tables 3 and 4. It is important to note that, although nine patients survived until the last examination day, the kinetic and kinematic data were not available from all of them: even though all patients were capable of walking unaided to the gait analysis laboratory (see supplementary video 2), once on the treadmill some of the animals refused to walk: Patient 7 simply lay down on the treadmill, and was finally enrolled only for performing the MR examinations and gathering the owners’ assessments. Patient 4 refused to walk on the treadmill before amputation; however, during the next sessions he walked perfectly, with valid trials. Other patients walked intermittently in such a way that the trials were not valid for analysis, even when they had previously walked perfectly on the treadmill. Moreover, although all owners were extremely cooperative, some were at times reluctant to allow their pets to be walked on the treadmill long enough to record valid trials.

Kinetic data showed that 10 days after amputation there was redistribution of the load to all remaining limbs, this load shift being more important towards the forelimbs (Figure 2 and Table 3). The values and pattern of load shifting are represented in Figure 2. The recorded PFz, MFz and IFz values showed no remarkable changes during the remaining examination time points, indicating that 10 days after the amputation the patient had already reached its new locomotory pattern. This was true for all patients including the lightest (8 kg) and the heaviest (54 kg) ones. Very interestingly, there were no statistically-significant differences between sessions (Table 3). With regard to the kinematic gait analysis, even though the patients walked smoothly on the treadmill (see supplementary video 1), there were significant differences between sessions in the means of almost all joint angles (Table 4). It is important to note that there were also important variations within a patient in the same session (not

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shown in Table 4). The mean joint angle progression curves showed a similar pattern between sessions (Figures 3 to 5), but they had huge individual variations (not shown).

Figure 2. Illustration of the load redistribution (LR).

LR averages for the A. peak (PFz); B. mean (MFz); and C. integral (IFz) values. The values in the bars indicate the mean % body weight (BW) loaded by each limb for each calculated parameter.

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40 Table 3. Results of the kinetic analysis

Pre 10 30 90 120 p

n = 10 n = 11 n = 10 n = 8 n = 8

PFz contralateral forelimb

Mean 64.88 71.45 67.69 69.78 66.74 0.2522

SD 7.00 4.73 8.71 6.44 8.27

PFz ipsilateral forelimb

Mean 67.78 71.97 70.86 70.64 74.18 0.7966

SD 9.03 5.90 9.29 5.60 11.56

PFz contralateral hind limb

Mean 47.60 55.68 54.44 53.96 58.75 0.4545

SD 11.71 10.07 9.62 13.69 15.36

MFz contralateral forelimb

Mean 47.55 52.71 49.33 50.70 48.49 0.3277

SD 7.13 4.71 6.84 4.37 7.45

MFz ipsilateral forelimb

Mean 47.10 50.27 50.25 48.30 47.53 0.8164

SD 6.93 5.30 7.79 3.43 9.55

MFz contralateral hind limb

Mean 33.37 35.41 35.70 35.08 38.09 0.8656

SD 7.02 6.27 6.42 10.38 10.48

IFz contralateral forelimb

Mean 30.28 32.09 30.41 30.07 28.82 0.9455

SD 7.28 7.46 9.10 8.45 7.54

IFz ipsilateral forelimb

Mean 28.20 28.09 28.61 26.08 28.10 0.9671

SD 5.90 7.12 6.57 6.31 6.81

IFz contralateral hind limb

Mean 21.45 22.65 23.81 21.51 23.21 0.9993

SD 7.28 7.00 8.19 5.23 7.62

p: p value of the Kruskal-Wallis test; PFz: Peak vertical forces; MFz: Mean values of the vertical forces; IFz:

Integral of the vertical forces; SD: Standard deviation

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Figure 3. Joint angle progression curves of each measured angle in the contralateral forelimb. Note the similarity of the curves before and after amputation.

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Figure 4. Joint angle progression curves of each measured angle in the ipsilateral forelimb.

Note the similarity of the curves before and after amputation.

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Figure 5. Joint angle progression curves of each measured angle in the contralateral hind limb. Note the similarity of the curves before and after amputation.

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Table 4. Results of the kinematic analysis

Pre (n=9) 10 (n=10) 30 (n=9) 90 (n=7) 120 (n=6) p

Contralateral scapulohumeral joint Mean ± SD 115.2 ± 5.67 117.9 ± 5.83 115.4 ± 5.54 112.7 ± 6.09 112.3 ± 4.24 <0.0001 ROM ± SD 27.68 ± 7.48 31.46 ± 8.12 29.51 ± 6.73 31.49 ± 6.26 26.07 ± 6.04 0.4784 Contralateral cubital joint Mean ± SD 114.9 ± 12.93 115.9 ± 12.82 126.5 ± 11.59 127.2 ± 13.67 123.6 ± 13.91 <0.0001

ROM ± SD 57.98 ± 15.56 61.8 ± 11.64 56.51 ± 14.74 60.75 ± 13.06 62.74 ± 9.60 0.8981 Contralateral carpal joint Mean ± SD 192.2 ± 26.85 191.5 ± 24.89 190.1 ± 26.03 191.5 ± 25.66 185.8 ± 26.88 <0.0001

ROM ± SD 99.38 ± 19.62 97.45 ± 11.28 104.7 ± 10.83 94.25 ± 13.65 101.1 ± 8.17 0.6661 Ipsilateral scapulohumeral joint Mean ± SD 117.5 ± 7.92 120.1 ± 8.33 111.9 ± 6.87 112.4 ± 6.94 112.1 ± 7.12 <0.0001

ROM ± SD 34.11 ± 5.69 32.2 ± 4.35 29.77 ± 4.80 32.17 ± 2.71 31.06 ± 5.03 0.6546 Ipsilateral cubital joint Mean ± SD 114.6 ± 12.28 117.6 ± 13.33 116.7 ± 12.94 121.5 ± 13.63 123.8 ± 13.58 <0.0001

ROM ± SD 55 ± 11.81 57.96 ± 11.92 57.23 ± 12.69 59.84 ± 12.73 57.78 ± 11.82 0.9168 Ipsilateral carpal joint Mean ± SD 185.5 ± 26.36 185.6 ± 24.54 190.6 ± 25.54 185.9 ± 23.23 185.5 ± 26.05 <0.0001

ROM ± SD 92.65 ± 19.14 91.83 ± 18.76 91.01 ± 15.71 82.42 ± 12.51 93.36 ± 10.05 0.6301 Contralateral coxofemoral joint Mean ± SD 116.1 ± 8.37 118.4 ± 6.96 120.8 ± 7.516 116.8 ± 6.29 116.1 ± 6.64 <0.0001

ROM ± SD 30.25 ± 6.22 27.47 ± 9.90 30.08 ± 9.71 25.17 ± 9.09 24.89 ± 6.27 0.5011 Contralateral femorotibial joint Mean ± SD 123.2 ± 7.48 115.8 ± 6.62 110.4 ± 5.76 114.7 ± 5.55 113.3 ± 5.68 <0.0001

ROM ± SD 42.4 ± 4.28 37.67 ± 8.44 37.82 ± 7.34 40.8 ± 12.16 34.83 ± 8.31 0.2990 Contralateral tarsal joint Mean ± SD 130.5 ± 8.98 121 ± 10.93 117.8 ± 14.07 124.9 ± 12.21 127.2 ± 10.75 <0.0001

ROM ± SD 47.74 ± 10.2 50.57 ± 10.52 61.27 ± 14.25 57.73 ± 11.24 48.04 ± 14.17 0.1432 Mean: mean joint angle calculated from the mean joint angle progression curves; SD: Standard deviation; ROM = Range of Motion; p = p value of the Kruskal-Wallis test

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Despite all these different kinematic results, ROMs of all analyzed joints were very similar before and after amputation and remained constant in the subsequent sessions after the amputation, without significant differences between sessions (Table 4).

3.4.3 MR evaluation of the contralateral stifle joint

Postoperative MR examination was possible only in eight patients. Although nine patients survived until the end of the study, severe metastatic disease was detected in patient 11 on day 120, and the MR examination was not performed. Postmortem MR examination was not possible in any case. No changes in the signal intensity of the CrCL, CdCL or the lateral and medial menisci were found, in comparison with the preoperative MR images. No changes in the cartilage surface, and no evidence of osteoarthritic changes were found (Figure 6).

Figure 6. Examples of MR images (sequence: PDW Vista Spair, sagittal plane).

A. before and B. 120 days after amputation. No changes could be detected in the joint 120 days after the procedure.

3.4.4 Owner evaluation of patient comfort

The owners’ assessment of patient comfort and the final questionnaire were made in German and translated into English as accurately as possible. The results of the owners’ assessment of

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patient comfort are presented in table 5 and show a clear tendency of the patients to improve after amputation. The patient numbers were entered in this Table, in order to enable the reader to see the individual outcome of each animal. Ten owners answered the final questionnaire:

nine from patients surviving until the end of the study and one from a patient which died the very same day of the final examination (Patient 6). This questionnaire revealed a high degree of owner satisfaction with the amputation result: Eight owners were very satisfied with the results of the amputation and two were satisfied; none were dissatisfied. Seven owners considered that the dog had adapted very well to the amputation, two that it had adapted well, and one that it had a fair outcome; none considered the outcome as poor. Seven owners considered that the dog took less time than expected to recover, three that recovery time was as expected and none considered that recovery took longer than expected. Seven owners responded that no behavioral changes had occurred and three gave affirmative answers: “now the dog is afraid of going upstairs, downstairs is no problem”, “when walking outside, the dog gets tired more easily” and one owner did not give any reason. Most (eight) owners were prevented from allowing the amputation to be performed: they feared that the animal could not move after the amputation and lead a normal life (4 cases), fear of the procedure itself (1 case), fear of behavioral changes (2 cases) and fear of a decrease in life quality (1 case). All owners would take the decision to amputate again, in case it is required in another dog. Two owners considered that the degree of activity after the amputation increased, six that it remained the same, and only two that it had decreased, although an owner stated that this was

"minimal". Seven owners considered that the life quality of the patient increased after the amputation, four that it remained the same and none considered that it had decreased.

Additional comments included: “we have not regretted the decision to amputate the dog for a second”, “we are happy we made the decision to have the dog amputated”, “I would advise other owners to allow the amputation. The need for amputation is no reason to euthanize a dog. The dog runs just as before surgery”, “The dog stands up without any difficulty”.

Especially remarkable were the comments of the owners of patient 6 (died just before the last session) “Thanks to the amputation we could enjoy the company of our dog for a few additional months. She was free of pain” and of patient 12 (bilateral hip osteoarthritis)

“Thanks for offering us the possibility to have our dog amputated. It was the right decision”.